This proposal targets a critical issue in the fight against the growing problem of antibiotic resistance: the search for strategies aimed at preserving the effectiveness of currently available antibiotics. Our model system is the aminoglycoside 6'-N-acetyltransferase type lb [AAC(6')-lb], an enzyme that mediates resistance to amikacin and other aminoglycosides. Our long term goal is to develop antisense oligonucleotides as pharmacological tools to selectively inhibit the expression of aac(6')-lb. To develop two kinds of antisense compounds that inhibit expression of aac(6')-lb by different mechanisms we designed specific aims 1 and 2: 1. Identification of nuclease-resistant oligodeoxynucleotide analogs that promote RNase H-mediated degradation of aac(6')-lb mRNA. We will design nuclease-resistant analogs and test their ability to mediate phenotypic conversion to amikacin susceptibility and determine the mechanism of action. 2. In vivo studies on RNase P-mediated degradation of aac(6')-lb mRNA by oligoribonucleotides and systematic analysis of the ability of nuclease-resistant oligoribonucleotide analogs to induce RNase P cleavage. We will design plasmids that code for selected oligoribonucleotides and test if they induce RNase P-mediated conversion to amikacin susceptibility. We will also carry out a systematic study on nuclease-resistant oligoribonucleotide analogs to determine which ones, if any, do not compromise RNase P-mediated cleavage of RNA. While achieving specific aims 1 and 2 will be an important step towards developing antisense compounds to preserve the efficacy of amikacin, many problems will remain to be solved. Two of these problems are: a) delivery methods to insure that antisense compounds reach the bacterial cell's cytoplasm are very limited; and b) the aac(6')-lb gene is often found in high copy number plasmids; as a consequence the large number of gene copies may make it very difficult to completely turn off expression. Specific aims 3 and 4 have been designed to deal with these problems: 3. Development of liposome formulations capable of delivering oligonucleotides into the cell's cytosol. We will test the ability of several formulations of cationic liposome-encapsulated oligonucleotide analogs to reach the cytoplasm. The process of internalization will be characterized by fluorescence microscopy. 4. Search for peptide inhibitors of the AAC(6')-lb enzyme. Enzyme inhibitors could have a synergistic activity with antisense oligonucleotides by inhibiting the action of any residual AAC(6')-lb protein synthesized. Peptide inhibitors will be searched using phage display.